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The effect of binding of divalent metal cations (Ca2+, Cu2+, Mg2+, Mn2+, Zn2+) on the kinetics of fibril formation of bovine α-lactalbumin at acidic conditions is considered. The kinetic parameters of the process were determined using a thioflavin T fluorescence assay. The DSC thermograms of bovine α-lactalbumin in the presence and absence of cations were recorded. The duration of the lag period correlates with the changes in the thermal stability of the molten globule of the protein in the presence of cations. The final thioflavin T fluorescence intensity after formation of the mature fibrils decreases under the influence of calcium ions which strongly bind to the monomeric protein, and increases in solutions containing copper and especially zinc. These ions seem to accelerate secondary nucleation processes and change the fibril morphology, which was confirmed by atomic force microscopy imaging. [ABSTRACT FROM AUTHOR]

The latex of the medicinal plant Artocarpus lakoocha (A. lakoocha), which has been shown to have potential anti-inflammatory and wound-healing capabilities, contains a novel heme-peroxidase. This protein was subjected to activity assays, fluorescence spectroscopy, and far-UV circular dichroism to investigate its structure, dynamics, and stability. The results demonstrated the presence of three folding states: the native state (N) at neutral pH, intermediate states including molten globule (MG) at pH 2 and acid-unfolded (UA) at pH 1.5 or lower, and acid-refolded (A) at pH 0.5, along with alkaline denatured (UB) at pH 8-12 and the third denatured state (D) at GuHCl concentrations exceeding 5 M. Absorbance studies indicated the presence of loosely associated form of heme in the pH range of 1-2. The protein showed stability and structural integrity across a wide pH range (3-10), temperature (70°C), and high concentrations of GuHCl (5 M) and urea (8 M). This study is the first to report multiple 'partially folded intermediate states' of A. lakoocha peroxidase, with varying amounts of secondary structure, stability, and compactness. These results demonstrate the high stability of A. lakoocha peroxidase and its potential for biotechnological and industrial applications, making it a valuable model system for further studies on its structure-function relationship. [ABSTRACT FROM AUTHOR]

The poly-reactivity of antibodies is defined as their binding to specific antigens as well as to related proteins and also to unrelated targets. Poly-reactivity can occur in individual molecules of natural serum antibodies, likely due to their conformation flexibility, and, for therapeutic antibodies, it plays a critical role in their clinical development. On the one hand, it can enhance their binding to target antigens and cognate receptors, but, on the other hand, it may lead to a loss of antibody function by binding to off-target proteins. Notably, poly-reactivity has been observed in antibodies subjected to treatments with dissociating, destabilizing or denaturing agents, in particular acidic pH, a common step in the therapeutic antibody production process involving the elution of Protein-A bound antibodies and viral clearance using low pH buffers. Additionally, poly-reactivity can emerge during the affinity maturation in the immune system, such as the germinal center. This review delves into the underlying potential causes of poly-reactivity, highlighting the importance of conformational flexibility, which can be further augmented by the acid denaturation of antibodies and the introduction of arginine mutations into the complementary regions of antibody-variable domains. The focus is placed on a particular antibody's acid conformation, meticulously characterized through circular dichroism, differential scanning calorimetry, and sedimentation velocity analyses. By gaining a deeper understanding of these mechanisms, we aim to shed light on the complexities of antibody poly-reactivity and its implications for therapeutic applications. [ABSTRACT FROM AUTHOR]

The latex of the medicinal plant Artocarpus lakoocha (A. lakoocha), which has been shown to have potential anti-inflammatory and wound-healing capabilities, contains a novel heme-peroxidase. This protein was subjected to activity assays, fluorescence spectroscopy, and far-UV circular dichroism to investigate its structure, dynamics, and stability. The results demonstrated the presence of three folding states: the native state (N) at neutral pH, intermediate states including molten globule (MG) at pH 2 and acid-unfolded (UA) at pH 1.5 or lower, and acid-refolded (A) at pH 0.5, along with alkaline denatured (UB) at pH 8-12 and the third denatured state (D) at GuHCl concentrations exceeding 5 M. Absorbance studies indicated the presence of loosely associated form of heme in the pH range of 1-2. The protein showed stability and structural integrity across a wide pH range (3-10), temperature (70°C), and high concentrations of GuHCl (5 M) and urea (8 M). This study is the first to report multiple ‘partially folded intermediate states’ of A. lakoocha peroxidase, with varying amounts of secondary structure, stability, and compactness. These results demonstrate the high stability of A. lakoocha peroxidase and its potential for biotechnological and industrial applications, making it a valuable model system for further studies on its structure-function relationship.

Disorder prediction methods that can discriminate between ordered and disordered regions have contributed fundamentally to our understanding of the properties and prevalence of intrinsically disordered proteins (IDPs) in proteomes as well as their functional roles. However, a recent large-scale assessment of the performance of these methods indicated that there is still room for further improvements, necessitating novel approaches to understand the strengths and weaknesses of individual methods. In this study, we compared two methods, IUPred and disorder prediction, based on the pLDDT scores derived from AlphaFold2 (AF2) models. We evaluated these methods using a dataset from the DisProt database, consisting of experimentally characterized disordered regions and subsets associated with diverse experimental methods and functions. IUPred and AF2 provided consistent predictions in 79% of cases for long disordered regions; however, for 15% of these cases, they both suggested order in disagreement with annotations. These discrepancies arose primarily due to weak experimental support, the presence of intermediate states, or context-dependent behavior, such as binding-induced transitions. Furthermore, AF2 tended to predict helical regions with high pLDDT scores within disordered segments, while IUPred had limitations in identifying linker regions. These results provide valuable insights into the inherent limitations and potential biases of disorder prediction methods. [ABSTRACT FROM AUTHOR]

The inherited disorder oculocutaneous albinism type 1 (OCA1) is caused by mutations in the TYR gene encoding tyrosinase (Tyr), an enzyme essential to producing pigments throughout the human body. The intramelanosomal domain of Tyr consists of the cysteine-rich and tyrosinase catalytic subdomains, which are essential for enzymatic activity. In protein unfolding, the roles of these subdomains are not well established. Here, we performed six molecular dynamics simulations at room temperature for Tyr and OCA1-related mutant variants P406L and R402Q intramelanosomal domains. The proteins were simulated for 1 μs in water and urea to induce unfolding. In urea, we observed increases in surface area, decreases in intramolecular hydrogen bonding, and decreases in hydrophobic interactions, suggesting a 'molten globule' state for each protein. Between all conditions, the cysteine-rich subdomain remains stable, whereas the catalytic subdomain shows increased flexibility. This flexibility is intensified by the P406L mutation, while R402Q increases the catalytic domain's rigidity. The cysteine-rich subdomain is rigid, preventing the protein from unfolding, whereas the flexibility of the catalytic subdomain accommodates mutational changes that could inhibit activity. These findings match the conclusions from our experimental work suggesting the function alteration by the P406L mutation, and the potential role of R402Q as a polymorphism. [ABSTRACT FROM AUTHOR]

"Newly Born" proteins, devoid of detectable homology to any other proteins, known as orphan proteins, occur in a single species or within a taxonomically restricted gene family. They are generated by the expression of novel open reading frames, and appear throughout evolution. We were curious if three recently developed programs for predicting protein structures, namely, AlphaFold2, RoseTTAFold, and ESMFold, might be of value for comparison of such "Newly Born" proteins to random polypeptides with amino acid content similar to that of native proteins, which have been called "Never Born" proteins. The programs were used to compare the structures of two sets of "Never Born" proteins that had been expressed—Group 1, which had been shown experimentally to possess substantial secondary structure, and Group 3, which had been shown to be intrinsically disordered. Overall, although the models generated were scored as being of low quality, they nevertheless revealed some general principles. Specifically, all four members of Group 1 were predicted to be compact by all three algorithms, in agreement with the experimental data, whereas the members of Group 3 were predicted to be very extended, as would be expected for intrinsically disordered proteins, again consistent with the experimental data. These predicted differences were shown to be statistically significant by comparing their accessible surface areas. The three programs were then used to predict the structures of three orphan proteins whose crystal structures had been solved, two of which display novel folds. Surprisingly, only for the protein which did not have a novel fold, and was taxonomically restricted, rather than being a true orphan, did all three algorithms predict very similar, high‐quality structures, closely resembling the crystal structure. Finally, they were used to predict the structures of seven orphan proteins with well‐identified biological functions, whose 3D structures are not known. Two proteins, which were predicted to be disordered based on their sequences, are predicted by all three structure algorithms to be extended structures. The other five were predicted to be compact structures with only two exceptions in the case of AlphaFold2. All three prediction algorithms make remarkably similar and high‐quality predictions for one large protein, HCO_11565, from a nematode. It is conjectured that this is due to many homologs in the taxonomically restricted family of which it is a member, and to the fact that the Dali server revealed several nonrelated proteins with similar folds. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:Proteins:3 [ABSTRACT FROM AUTHOR]

Pulsed electric field (PEF) is an effective way to modulate the structure and activity of enzymes; however, the dynamic changes in enzyme structure during this process, especially the intermediate state, remain unclear. In this study, the molten globule (MG) state of α-amylase under PEF processing was investigated using intrinsic fluorescence, surface hydrophobicity, circular dichroism, etc. Meanwhile, the influence of coexisting carrageenan on the structural transition of α-amylase during PEF processing was evaluated. When the electric field strength was 20 kV/cm, α-amylase showed the unique characteristics of an MG state, which retained the secondary structure, changed the tertiary structure, and increased surface hydrophobicity (from 240 to 640). The addition of carrageenan effectively protected the enzyme activity of α-amylase during PEF treatment. When the mixed ratio of α-amylase to carrageenan was 10:1, they formed electrostatic complexes with a size of ~20 nm, and carrageenan inhibited the increase in surface hydrophobicity (<600) and aggregation (<40 nm) of α-amylase after five cycles of PEF treatment. This work clarifies the influence of co-existing polysaccharides on the intermediate state of proteins during PEF treatment and provides a strategy to modulate protein structure by adding polysaccharides during food processing. [ABSTRACT FROM AUTHOR]

Molten globule (MG) is the name given to a compact, non‐native conformation of proteins that has stimulated the imagination and work in the protein folding field for more than 40 years. The MG has been proposed to play a central role in the folding reaction and in important cell functions, and to be related to the onset of misfolding diseases. Due to its inherent intractability to high‐resolution studies, atomistic structural models have not yet been obtained. We present here an integrative atomistic model of the MG formed at acidic pH by the apoflavodoxin from the human pathogen Helicobacter pylori. This MG has been previously shown to exhibit the archetypical expansion, spectroscopic and thermodynamic features of a molten conformation. To obtain the model, we have analyzed the stability of wild‐type and 55 apoflavodoxin mutants to derive experimental equilibrium Φ values that have been used in biased molecular dynamics simulations to convert the native conformation into an MG ensemble. The ensemble has been refined to reproduce the experimental hydrodynamic radius and circular dichroism (CD) spectrum. The refined ensemble, deposited in PDB‐Dev, successfully explains the characteristic 1H‐nuclear magnetic resonance (NMR) and near‐UV CD spectral features of the MG as well as its solvent‐accessible surface area (SASA) change upon unfolding. This integrative model of an MG will help to understand the energetics and roles of these elusive conformations in protein folding and misfolding. Interestingly, the apoflavodoxin MG is structurally unrelated to previously described partly unfolded conformations of this protein, exemplifying that equilibrium MGs need not to reflect the properties of kinetic intermediates. PDB Code(s): PDBDEV_00000112; [ABSTRACT FROM AUTHOR]

The poly-reactivity of antibodies is defined as their binding to specific antigens as well as to related proteins and also to unrelated targets. Poly-reactivity can occur in individual molecules of natural serum antibodies, likely due to their conformation flexibility, and, for therapeutic antibodies, it plays a critical role in their clinical development. On the one hand, it can enhance their binding to target antigens and cognate receptors, but, on the other hand, it may lead to a loss of antibody function by binding to off-target proteins. Notably, poly-reactivity has been observed in antibodies subjected to treatments with dissociating, destabilizing or denaturing agents, in particular acidic pH, a common step in the therapeutic antibody production process involving the elution of Protein-A bound antibodies and viral clearance using low pH buffers. Additionally, poly-reactivity can emerge during the affinity maturation in the immune system, such as the germinal center. This review delves into the underlying potential causes of poly-reactivity, highlighting the importance of conformational flexibility, which can be further augmented by the acid denaturation of antibodies and the introduction of arginine mutations into the complementary regions of antibody-variable domains. The focus is placed on a particular antibody’s acid conformation, meticulously characterized through circular dichroism, differential scanning calorimetry, and sedimentation velocity analyses. By gaining a deeper understanding of these mechanisms, we aim to shed light on the complexities of antibody poly-reactivity and its implications for therapeutic applications.

The inherited disorder oculocutaneous albinism type 1 (OCA1) is caused by mutations in the TYR gene encoding tyrosinase (Tyr), an enzyme essential to producing pigments throughout the human body. The intramelanosomal domain of Tyr consists of the cysteine-rich and tyrosinase catalytic subdomains, which are essential for enzymatic activity. In protein unfolding, the roles of these subdomains are not well established. Here, we performed six molecular dynamics simulations at room temperature for Tyr and OCA1-related mutant variants P406L and R402Q intramelanosomal domains. The proteins were simulated for 1 μs in water and urea to induce unfolding. In urea, we observed increases in surface area, decreases in intramolecular hydrogen bonding, and decreases in hydrophobic interactions, suggesting a ‘molten globule’ state for each protein. Between all conditions, the cysteine-rich subdomain remains stable, whereas the catalytic subdomain shows increased flexibility. This flexibility is intensified by the P406L mutation, while R402Q increases the catalytic domain’s rigidity. The cysteine-rich subdomain is rigid, preventing the protein from unfolding, whereas the flexibility of the catalytic subdomain accommodates mutational changes that could inhibit activity. These findings match the conclusions from our experimental work suggesting the function alteration by the P406L mutation, and the potential role of R402Q as a polymorphism.

The functionality of the tumor suppressor p53 is altered in more than 50% of human cancers, and many individuals with cancer exhibit amyloid-like buildups of aggregated p53. An understanding of what triggers the pathogenic amyloid conversion of p53 is required for the further development of cancer therapies. Here, perturbation of the p53 core domain (p53C) with subdenaturing concentrations of guanidine hydrochloride and high hydrostatic pressure revealed native-like molten globule (MG) states, a subset of which were highly prone to amyloidogenic aggregation. We found that MG conformers of p53C, probably representing population-weighted averages of multiple states, have different volumetric properties, as determined by pressure perturbation and size-exclusion chromatography. We also found that they bind the fluorescent dye 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) and have a native-like tertiary structure that occludes the single Trp residue in p53. Fluorescence experiments revealed conformational changes of the single Trp and Tyr residues before p53 unfolding and the presence of MG conformers, some of which were highly prone to aggregation. p53C exhibited marginal unfolding cooperativity, which could be modulated from unfolding to aggregation pathways with chemical or physical forces. We conclude that trapping amyloid precursor states in solution is a promising approach for understanding p53 aggregation in cancer. Our findings support the use of single-Trp fluorescence as a probe for evaluating p53 stability, effects of mutations, and the efficacy of therapeutics designed to stabilize p53.

Proteins are generally resistant to large conformational changes under physiological conditions. Here, we show that platinum (Pt(II)), which is widely-used metal centre in cancer therapeutic drugs, binds to a cytosolic protein, small ubiquitin-like modifier 1 (SUMO1), under physiological conditions and changes its conformation to a molten globule (MG). Mass spectrometry (ICP-MS) studies confirmed stoichiometric Pt(II) binding to SUMO1. Fluorescence spectroscopy showed Tyr fluorescence quenching and increased ANS binding. Fluorescence assays on Trp-mutants indicated conformational changes and circular dichroism (CD) suggested MG formation upon Pt(II) binding. In contrast, structural homologues of SUMO1 (ubiquitin (Ubq) and SUMO2) showed no conformational changes on Pt(II) titration. Further studies compared the impact of distinct His residues in SUMO1 on Pt(II) binding and protein structure to SUMO2 and Ubq. Experiments at low pH (5.0) implicated His residues interacting with Pt(II), corroborated by the absence of conformational change in the H75L mutant of SUMO1. Pt(II)-His binding in SUMO1 unravels key molecular determinants of Pt(II)-protein interactions and their conformational consequences. SUMO1 with other SUMOylation components are shown to have enhanced expression in several cancers, hence, our study suggests a possible fate of the non-targetability of Pt(II)-based drugs on SUMOylation in cancer cells, upon interaction with  SUMO1., (© 2024 Wiley‐VCH GmbH.)

Bacteriorhodopsin (bR) of purple membrane (PM) has increasing technical interests, particularly in photonic devices and bioelectronics. The present work has concerned with monitoring the temperature dependence of passive electric responses in-plane and out-of-plane of the membranes. Based on thermal properties observed orthogonally here for PM, a high-temperature intermediate of bR has been suggested to populate at around 60 °C, which may be ascribed to a molten globule-like state. This intermediate has been found to be enclosed between two reversible thermal transitions for PM. Large-scale turnover in the energy of activation, for these two thermal transitions, occurs steeply at such state at 60 °C, above which does bR reverse the sign of dielectric anisotropy (i.e. crossover) provided the operating frequency should be above the crossover frequency, at which the reversal occurs. No such crossover was found to occur below the crossover frequency, even above the crossover temperature (i.e. 60 °C). Likewise, no such crossover was found to occur below the crossover temperature, even above the crossover frequency. Relying on this reasoning, a logic gate operation may be declared implicating bR for bioelectronics and sense technological relevance. In addition, the results specify "dual frequency" as well as "dual temperature" characteristics to bacteriorhodopsin., (© 2024 Wiley‐VCH GmbH.)

Quite a long time ago, Oleg B. Ptitsyn put forward a hypothesis about the possible functional significance of the molten globule (MG) state for the functioning of proteins. MG is an intermediate between the unfolded and the native state of a protein. Its experimental detection and investigation in a cell are extremely difficult. In the last decades, intensive studies have demonstrated that the MG-like state of some globular proteins arises from either their modifications or interactions with protein partners or other cell components. This review summarizes such reports. In many cases, MG was evidenced to be functionally important. Thus, the MG state is quite common for functional cellular proteins. This supports Ptitsyn's hypothesis that some globular proteins may switch between two active states, rigid (N) and soft (MG), to work in solution or interact with partners. [ABSTRACT FROM AUTHOR]

MERS-CoV main protease (Mpro) is essential for the maturation of the coronavirus; therefore, considered a potential drug target. Detailed conformational information is essential to developing antiviral therapeutics. However, the conformation of MERS-CoV Mpro under different conditions is poorly characterized. In this study, MERS-CoV Mpro was recombinantly produced in E.coli and characterized its structural stability with respect to changes in pH and temperatures. The intrinsic and extrinsic fluorescence measurements revealed that MERS-CoV Mpro tertiary structure was exposed to the polar environment due to the unfolding of the tertiary structure. However, the secondary structure of MERS-CoV Mpro was gained at low pH because of charge-charge repulsion. Furthermore, differential scanning fluorometry studies of Mpro showed a single thermal transition at all pHs except at pH 2.0; no transitions were observed. The data from the spectroscopic studies suggest that the MERS-CoV Mpro forms a molten globule-like state at pH 2.0. Insilico studies showed that the covid-19 Mpro shows 96.08% and 50.65% similarity to that of SARS-CoV Mpro and MERS-CoV Mpro, respectively. This study provides a basic understanding of the thermodynamic and structural properties of MERS-CoV Mpro. [ABSTRACT FROM AUTHOR]

• Thermostable serine protease showed a maximum intrinsic fluorescence in 335 nm and a blue shift in 510 nm for extrinsic fluorescence. • It was suggested that the protein transitions probably to an MG state at pH 3. • Thermostable serine protease showed a little shift in the MG state intermediate at pH 3 in far-UV. • Maximum decreases was observed in 228 to 300 nm for UV-near CD. Proteins in the molten globule (MG) stage have a reasonably stable secondary structure content, but changes in side-chain packing cause a noticeable decrease in the number of tertiary structures. Here, we investigated serine protease as a suitable model to study the folding route and characteristics of a protein in the MG state. Upon induction with IPTG, the protein exhibited a molecular weight of 42 kDa and achieved a final concentration of 1.7 mg/mL after the concentration process. Serine protease's intrinsic and extrinsic fluorescence was examined in the pH range of 1–12 using fluorescence spectroscopy with maximum intrinsic fluorescence in 335 nm and a blue shift in 510 nm for extrinsic fluorescence. It was suggested that the protein transitions probably to an MG state at pH 3. Evidences showed that serine protease's secondary structure using far-UV circular dichroism (CD) showed a little shift in the MG state intermediate, pH 3, relative to the original state, pH 8, which is typical for MG. Maximum decreases was observed in 228 to 300 nm for UV-near CD. Also revealed a notable alteration in the pH 3 of the enzyme compared to pH 8, which is the pH at which the protein takes on its natural structure. By using acrylamide to quench the fluorescence, the Stern-Volmer constant (KSV) was determined at pH values of 8 and 3, yielding values of 20 and 14 M −1, respectively. This is likely correlated with an increase in the enzyme's molecular volume in the MG state. Molecular dynamics (MD) simulation showed that although the thermal stability of this enzyme at 90° was confirmed in the Root-Mean-Square-Deviation (RMSD), Root-Mean-Square-Fluctuation (RMSF), Radius of gyration (Rg) as well as Solvent-Accessible-Surface-Area (SASA) graphs, but pH 3 may significantly disturb the tertiary structure and secondary structure. These in-silico results may also be analyzed in line with the occurrence of MG at pH 3. [ABSTRACT FROM AUTHOR]

[Display omitted] • Effect of non-native proline isomers on protein folding from pressure-jump NMR. • Accelerated proline peptide bond isomerization under protein folding conditions. • Molten globule characteristics for chains with non-native proline isomers. • Activation energy for Pro isomerization can be depressed under folding conditions. • Pressure lowers proline isomerization rates without impacting cis – trans equilibria. Proline isomerization is widely recognized as a kinetic bottleneck in protein folding, amplified for proteins rich in Pro residues. We introduced repeated hydrostatic pressure jumps between native and pressure-denaturing conditions inside an NMR sample cell to study proline isomerization in the pressure-sensitized L50A ubiquitin mutant. Whereas in two unfolded heptapeptides, X-Pro peptide bonds isomerized ca 1.6-fold faster at 1 bar than at 2.5 kbar, for ubiquitin ca eight-fold faster isomerization was observed for Pro-38 and ca two-fold for Pro-19 and Pro-37 relative to rates measured in the pressure-denatured state. Activation energies for isomerization in pressure-denatured ubiquitin were close to literature values of 20 kcal/mole for denatured polypeptides but showed a substantial drop to 12.7 kcal/mole for Pro-38 at atmospheric pressure. For ubiquitin isomers with a cis E18-P19 peptide bond, the 1-bar NMR spectrum showed sharp resonances with near random coil chemical shifts for the C-terminal half of the protein, characteristic of an unfolded chain, while most of the N-terminal residues were invisible due to exchange broadening, pointing to a metastable partially folded state for this previously recognized 'folding nucleus'. For cis -P37 isomers, a drop in pressure resulted in the rapid loss of nearly all unfolded-state NMR resonances, while the recovery of native state intensity revealed a slow component attributed to cis → trans isomerization of P37. This result implies that the NMR-invisible cis -P37 isomer adopts a molten globule state that encompasses the entire length of the ubiquitin chain, suggestive of a structure that mostly resembles the folded state. [ABSTRACT FROM AUTHOR]

Eps15 homology (EH) domains are universal interaction domains to establish networks of protein–protein interactions in the cell. These networks mainly coordinate cellular functions including endocytosis, actin remodeling, and other intracellular signaling pathways. They are well characterized in structural terms, except for the internal EH domain from human γ‐synergin (EHγ). Here, we complete the family of EH domain structures by determining the solution structure of the EHγ domain. The structural ensemble follows the canonical EH domain fold and the identified binding site is similar to other known EH domains. But EHγ differs significantly in the N‐ and C‐terminal regions. The N‐terminal α‐helix is shortened compared to known homologues, while the C‐terminal one is fully formed. A significant proportion of the remaining N‐ and C‐terminal regions are well structured, a feature not seen in other EH domains. Single mutations in both the N‐terminal and the C‐terminal structured extensions lead to the loss of the distinct three‐dimensional fold and turn EHγ into a molten globule like state. Therefore, we propose that the structural extensions in EHγ function as a clamp and are undoubtedly required to maintain its tertiary fold. PDB Code(s): 2MX7; [ABSTRACT FROM AUTHOR]

Understanding how proteins fold into their native structure is a fundamental problem in biophysics, crucial for protein design. It has been hypothesized that the formation of a molten globule intermediate precedes folding to the native conformation of globular proteins; however, its thermodynamic properties are poorly known. We perform singlemolecule pulling experiments of protein barnase in the range of 7 C to 37 C using a temperature-jump optical trap. We derive the folding free energy, entropy and enthalpy, and heat capacity change (Cp = 1,050 ± 50 cal/mol·K) at low ionic strength conditions. From the measured unfolding and folding kinetic rates, we also determine the thermodynamic properties of the transition state, finding a significant change in sCp (~90%) between the unfolded and the transition states. In contrast, the major change in enthalpy (~80%) occurs between the transition and native states. These results highlight a transition state of high energy and low configurational entropy structurally similar to the native state, in agreement with the molten globule hypothesis. [ABSTRACT FROM AUTHOR]

Pulsed electric field (PEF) is an effective way to modulate the structure and activity of enzymes; however, the dynamic changes in enzyme structure during this process, especially the intermediate state, remain unclear. In this study, the molten globule (MG) state of α-amylase under PEF processing was investigated using intrinsic fluorescence, surface hydrophobicity, circular dichroism, etc. Meanwhile, the influence of coexisting carrageenan on the structural transition of α-amylase during PEF processing was evaluated. When the electric field strength was 20 kV/cm, α-amylase showed the unique characteristics of an MG state, which retained the secondary structure, changed the tertiary structure, and increased surface hydrophobicity (from 240 to 640). The addition of carrageenan effectively protected the enzyme activity of α-amylase during PEF treatment. When the mixed ratio of α-amylase to carrageenan was 10:1, they formed electrostatic complexes with a size of ~20 nm, and carrageenan inhibited the increase in surface hydrophobicity (

An enigmatic and never described hyper‐reactivity of most of the cysteines resident in the reduced, molten globule‐like intermediate of a few proteins has been recently discovered. In particular, all ten cysteines of chymotrypsinogen showed hundred times increased reactivity against hydrophobic reagents. A single cysteine (Cys1) was also found thousand times more reactive toward GSSG, making speculate that a single glutathionylation could represent the primordial event of its oxidative folding. In the present study, we compare these kinetic properties with those present in trypsinogen taken in its reduced, molten globule‐like intermediate and identify the origin of these unusual properties. Despite the divergent evolution of these two proteins, the different amount of disulfides and the very different 3D localization of three disulfides, their hyper‐reactivity toward hydrophobic thiol reagents and disulfides is very similar. Mass spectrometry identifies two cysteines in trypsinogen, Cys148 and Cys197, 800 times more reactive toward GSSG than an unperturbed protein cysteine. These results point toward a stringent and accurate preservation of these peculiar kinetic properties during a divergent evolution suggesting some important role, which at the present can only be hypothesized. Similar extraordinary hyper‐reactivity has been found also in albumin, ribonuclease, and lysozyme confirming that it cannot be considered a kinetic singularity of a single protein. Interestingly, the very flexible and fluctuating structures like those typical of the molten globule status prove capable of enabling sophisticated actions typical of enzymes such as binding to GSSG with relevant specificity and high affinity (KD = 0.4 mm) and accelerating the reaction of its cysteines by thousands of times. [ABSTRACT FROM AUTHOR]

Microsecond timescale explicit-solvent atomistic simulations were carried out to investigate how anionic surfactants modulate protein structure and dynamics. We found that lysozyme undergoes near-complete denaturation at the high concentration (> 0.1 M) of sodium pentadecyl sulfonate (SPDS), while only partial denaturation occurs at the concentration slightly below 0.1 M. In large part, protein denaturation is structurally manifested by disappearance of helical segments and loss of tertiary interactions. The computational prediction of the extent of burial of cysteine residues was experimentally validated by measuring the accessibility of the respective sulfhydryl groups. Overall, our work indicates an interesting synergy between electrostatic and hydrophobic contributions to lysozyme's denaturation process by anionic surfactants. In fact, first disulfide bridges and hydrogen bonds from protein surface to SPDS head groups loosen the protein globule followed by fuller denaturation via insertion of the surfactant's hydrophobic tails into the protein core. [ABSTRACT FROM AUTHOR]

Quite a long time ago, Oleg B. Ptitsyn put forward a hypothesis about the possible functional significance of the molten globule (MG) state for the functioning of proteins. MG is an intermediate between the unfolded and the native state of a protein. Its experimental detection and investigation in a cell are extremely difficult. In the last decades, intensive studies have demonstrated that the MG-like state of some globular proteins arises from either their modifications or interactions with protein partners or other cell components. This review summarizes such reports. In many cases, MG was evidenced to be functionally important. Thus, the MG state is quite common for functional cellular proteins. This supports Ptitsyn’s hypothesis that some globular proteins may switch between two active states, rigid (N) and soft (MG), to work in solution or interact with partners.

The interior of the cell is crowded with different kinds of biological molecules with varying sizes, shapes and compositions which may affect physiological processes especially protein folding, protein conformation and protein stability. To understand the consequences of such a crowded environment, pH-induced unfolding of holo alpha-lactalbumin (holo α-LA) was studied in the presence of ethylene glycol (EG). The effect of EG on the folding and stability of holo α-LA in aqueous solution was investigated using several spectroscopic techniques. The results indicate that stabilization/destabilization of holo α-LA by EG is concentration- and pH-dependent. Low concentration of EG stabilizes the protein at pH near its pI. From the results of far-UV CD, UV–visible and ANS fluorescence, intermediate state (MG state) was characterized in the presence of high concentration of ethylene glycol. The results invoke a new mechanism for the formation of MG state identical to active component of BAMLET. MG state of holo α-LA has a direct implication to cancer therapy. MG state of α-LA in complex with specific type of lipid is a novel class of protein-based anti-cancer complexes that incorporate oleic acid and deliver it to the cancer cells. [ABSTRACT FROM AUTHOR]

We demonstrate that a molten globule‐like (MG) state of a protein, usually described as a compact yet non‐folded conformation that is only present in a narrow and delicate parameter range, is preserved in the high concentration environment of the protein hydrogel. We reveal mainly by means of electron paramagnetic resonance (EPR) spectroscopy that bovine serum albumin (BSA) retains the known basic MG state after a hydrogel has been formed from 20 wt% precursor solutions. At pH values of ~11.4, BSA hydrogels made from MG‐BSA remain stable for weeks, while gels formed at slightly different (~0.2) pH units above and below the MG‐state value dissolve into viscous solutions. On the contrary, when hydrophobic screening agents are added such as amphiphilic, EPR‐active stearic acid derivatives (16‐DOXYL‐stearic acid, 16‐DSA), the MG‐state based hydrogel is the least long‐lived, as the hydrophobic interaction of delicately exposed hydrophobic patches of BSA molecules is screened by the amphiphilic molecules. These bio‐ and polymer‐physically unexpected findings may lead to new bio‐compatible MG‐based hydrogels that display novel properties in comparison to conventional gels. [ABSTRACT FROM AUTHOR]

Apomyoglobin is a widely used model for studying the molecular mechanisms of globular protein folding. This work aimed to analyze the effects of rigidity and length of loops linking protein secondary structure elements on the stability of the molten globule intermediate state. For this purpose, we studied folding/unfolding of mutant apomyoglobin forms with substitutions of loop‐located proline residues to glycine and with loop extension by three or six glycine residues. The kinetic and equilibrium experiments performed gave an opportunity to calculate free energies of different apomyoglobin states. Our analysis revealed that the mutations introduced into the apomyoglobin loops have a noticeable effect on the stability of the intermediate state compared to the unfolded state. [ABSTRACT FROM AUTHOR]

The influence of a wide spectrum of water-miscible organic cosolvents at different concentrations on the denaturation of hen egg-white lysozyme is studied using differential scanning calorimetry (DSC) and circular dichroism (CD). The denaturing ability of cosolvents is characterized with the parameter − ∂ T d ∂ x 1 reflecting the change in the denaturation temperature with increasing cosolvent concentration. A series of cosolvents according to their denaturing ability is established: glycerol < ethylene glycol < pure water < dimethyl sulfoxide < methanol < ethanol < formamide < acetonitrile, dimethyl formamide, acetone < 2-propanol < 1,4-dioxane < tert-butanol < 1-propanol < tetrahydrofuran < 2-butanol < 1-butanol. The link of the − ∂ T d ∂ x 1 parameter to the m values obtained in isothermal studies of chemically induced denaturation and to the solvation properties of aqueous-organic mixtures is demonstrated. Near-UV CD measurements indicate that changes in the tertiary structure occur at slightly lower temperature than the DSC peak in some of the mixtures with high organic cosolvent content. Far-UV CD measurements in the mixtures containing alcohols or tetrahydrofuran confirm non-simultaneous disruption of the tertiary and secondary lysozyme structure. Organic cosolvents induce formation of the molten globule state with preserved and even increased secondary structure, which gradually disrupts at higher temperatures. • A series of water-miscible organic cosolvents by their protein denaturing ability is established. • DSC peak and near-UV signal jump can occur at different temperatures in aqueous-organic mixtures. • Secondary structure keeps while tertiary is disrupted at T d. • Denaturing ability of cosolvents correlates with solvation properties of the mixtures. [ABSTRACT FROM AUTHOR]

The Golgi complex is a central component of the secretory pathway, responsible for several critical cellular functions in eukaryotes. The complex is organized by the Golgi matrix that includes the Golgi reassembly and stacking protein (GRASP), which was shown to be involved in cisternae stacking and lateral linkage in metazoan. GRASPs also have critical roles in other processes, with an unusual ability to interact with several different binding partners. The conserved N terminus of the GRASP family includes two PSD‐95, DLG, and ZO‐1 (PDZ) domains. Previous crystallographic studies of orthologues suggest that PDZ1 and PDZ2 have similar conformations and secondary structure content. However, PDZ1 alone mediates nearly all interactions between GRASPs and their partners. In this work, NMR, synchrotron radiation CD, and molecular dynamics (MD) were used to examine the structure, flexibility, and stability of the two constituent PDZ domains. GRASP PDZs are structured in an unusual β3α1β4β5α2β6β1β2 secondary structural arrangement and NMR data indicate that the PDZ1 binding pocket is formed by a stable β2‐strand and a more flexible and unstable α2‐helix, suggesting an explanation for the higher PDZ1 promiscuity. The conformational free energy profiles of the two PDZ domains were calculated using MD simulations. The data suggest that, after binding, the protein partner significantly reduces the conformational space that GRASPs can access by stabilizing one particular conformation, in a partner‐dependent fashion. The structural flexibility of PDZ1, modulated by PDZ2, and the coupled, coordinated movement between the two PDZs enable GRASPs to interact with multiple partners, allowing them to function as promiscuous, multitasking proteins. [ABSTRACT FROM AUTHOR]

To understand protein folding problem under physiological condition, usually taken as dilute aqueous buffer at pH 7.0 and 25 °C, knowledge of properties of folding intermediates is important, such as molten globule (MG). We observed that polyethylene glycol 400 Da (PEG 400) induces molten globule state conformation in cytochrome c at pH 7.0 and 25 °C. This PEG-induced MG state has: (i) native tertiary structure partially perturbed, (ii) unperturbed native secondary structure, (iii) newly exposed hydrophobic patches, and (iv) has 1.58 times more hydrodynamic volume than that of the native protein. Isothermal titration calorimetry and docking studies showed specific binding between PEG 400 and cytochrome c. The study delineates that PEG-protein interactions are more complex than the excluded-volume. The soft interactions need to be seriously studied in crowding milieu that leads to destabilization of protein and overcome stabilizing exclusion volume effect. This study not only can help in unraveling the mystery of steps involved in the proper folding of proteins to solve the massively complicated problems of protein folding but also provides novel insights towards importance of structural change in proteins inside cell where intermediate states of protein import-export easily via membranes rather than native form of proteins. Unlabelled Image • PEG 400 is a low-level toxic ingredient in drug formulations to augment the solubility and bioavailability of weakly water-soluble drugs. • The PEG 400 yields molten globule of holo-cytochrome c , characterized at physiological pH and 25 oC. • The PEG 400 interacts with cytochrome c and its prosthetic group by soft interactions that lead the structural change in the protein. • The exclusion volume effect is surmounted by soft interactions which leads formation of stabilized state of cytochrome c (molten globule). • Importance of formation of molten globule lies in protein import-export easily via membranes rather than native form of proteins. [ABSTRACT FROM AUTHOR]

Proteins fold via a number of intermediates that help them to attain their unique native 3D structure. These intermediates can be trapped under extreme conditions of pH, temperature and chemical denaturants. Similar states can also be achieved by other processes like chemical modification, site directed mutagenesis (or point mutation) and cleavage of covalent bonds of natural proteins under physiological conditions usually taken as dilute buffer (near neutral pH) and 25 °C. Structural characterization of molten globules is hampered due to (i) their transient nature, (ii) very low population at equilibrium, and (iii) prone to aggregation at high concentration. Furthermore, the dynamic nature of these folding intermediates makes them unsuitable for X-ray diffraction. Hence, understanding their structures at the atomic level is often a challenge. However, characterization of these intermediates at the atomic level is possible by NMR, which could possibly unravel new details of the protein folding process. We have previously shown that the L94G mutant of horse cytochrome-c displays characteristics of the molten globule (MG) state at pH 6.0 and 25 °C. As a first step towards characterizing this MG state at the atomic level by NMR, we report its complete backbone, side chain and heme chemical shift assignments. [ABSTRACT FROM AUTHOR]

Ovalbumin (OVA) is widely used to prepare nanocomposites for the delivery of bioactive compounds. During long-term storage of shell eggs, natural OVA is slowly transformed into a more heat-stable form called S-OVA, which can also be obtained artificially through alkali-heat treatment. This study aimed to reveal the effects of alkali-heat and ultrasonic treatment on the structure, stability and polyphenol encapsulation ability of OVA by analyzing the interaction mechanism through molecular docking and multispectral analysis. The results showed that the proteins treated with alkali-heat and ultrasound had higher stability and surface hydrophobicity, reflecting the change in their functional properties. The S-OVA generated by alkali-heat treatment had higher thermal stability and formed softer gels than those generated by ultrasound treatment. The results of infrared spectroscopy and molecular docking indicated that hydrophobic interactions and hydrogen bonds were the main forces stabilizing the formation of catechin-OVA complexes. X-ray diffraction showed that OVA was a good carrier for catechins, which existed in an amorphous state in the hydrophobic core of proteins. Both ultrasound and alkali-heat treatments exposed hydrophobic groups in the protein and significantly enhanced the ability of the protein to bind polyphenols (increased from 15.72 nmol/mg to 44.10 nmol/mg and 43.18 nmol/mg, respectively), resulting in greater antioxidant capacity. This study mainly revealed the binding mode of OVA with polyphenols and explained the reasons for the changes in the functional properties of OVA by revealing the structural changes in proteins under different treatments, providing a basis for the advantages of alkali-heat and ultrasonic treatment of OVA in the food industry. [Display omitted] • The effects of alkali-heat and ultrasound treatment on the OVA were verified. • Alkali-heat treatment improved the thermal stability of OVA. • Alkali-heat and ultrasound treatment of OVA enhanced its functional properties. • Catechin-OVA complex was dominated by hydrogen bonds and hydrophobic interactions. [ABSTRACT FROM AUTHOR]

The foaming properties of egg white protein (EWP) after pH-induced unfolding (pH 2, pH 4, pH 10 and pH 12) and refolding treatment (pH 2–7, pH 4–7, pH 10–7 and pH 12-7 (adjusting the above pH back to 7)) were studied by structure changes and quantitative proteomic analysis. It was found that all pH treatment improved foaming capacity (FC) and foaming stability (FS) of EWP, especially at pH 2 and pH 12, where EWP was in a molten globule state with unfolded structure (especially at pH 12), high surface hydrophobicity (except for pH 12-treated EWP), more free sulfhydryl groups and up-regulated adsorption proteins. However, adjusting the pH back to 7 limited the foaming properties due to protein refolding. Meanwhile the presence of down-regulated proteins was another reason for relatively poor FC at pH 2–7. Notably, the firmest foam and high FS at pH 12-7 were attributed to the generation of aggregates and the long-term stability of adsorbed proteins. The results of quantitative proteomics implied that lysozyme, ovalbumin and protein TENP were identified as key proteins associated with excellent FC at pH 2, pH 12 and pH 12-7, while β-ovomucin contributed the most to FC at pH 2–7. Moreover, protein TENP and ovomucoid were more relevant to maintaining and reducing FS. Overall, this work elucidated the mechanism of pH treatment to enhance the foaming properties of EWP, which would provide the necessary theoretical support for the quality improvement and product development of EWP powder. [Display omitted] • Molten globule structure of EWP at pH 2 and pH 12 markedly improved foaming ability. • Adjusting the pH back to 7 limited the foaming properties due to protein refolding. • The foams of pH-induced EWP were firmer, especially at pH 12-7. • The presence of up-regulated proteins explained high FC at pH 2, pH 12 and pH 12-7. • Protein TENP contributed more to FS, while ovomucoid had a negative effect on FS. [ABSTRACT FROM AUTHOR]

Gram-positive bacteria are ancient organisms. Many bacteria, including Gram-positive bacteria, produce toxins to manipulate the host, leading to various diseases. While the targets of Gram-positive bacterial toxins are diverse, many of those toxins use a similar mechanism to invade host cells and exert their functions. Clostridial neurotoxins produced by Clostridial tetani and Clostridial botulinum provide a classical example to illustrate the structure–function relationship of bacterial toxins. Here, we critically review the recent progress of the structure–function relationship of clostridial neurotoxins, including the diversity of the clostridial neurotoxins, the mode of actions, and the flexible structures required for the activation of toxins. The mechanism clostridial neurotoxins use for triggering their activity is shared with many other Gram-positive bacterial toxins, especially molten globule-type structures. This review also summarizes the implications of the molten globule-type flexible structures to other Gram-positive bacterial toxins. Understanding these highly dynamic flexible structures in solution and their role in the function of bacterial toxins not only fills in the missing link of the high-resolution structures from X-ray crystallography but also provides vital information for better designing antidotes against those toxins.

The initial steps of the folding pathway of the C-terminal domain of the murine prion protein mPrP(90–231) are predicted based on the sequential collapse model (SCM). A non-local dominant contact is found to form between the connecting region between helix 1 and β-sheet 1 and the C-terminal region of helix 3. This non-local contact nucleates the most populated molten globule-like intermediate along the folding pathway. A less stable early non-local contact between segments 120–124 and 179–183, located in the middle of helix 2, promotes the formation of a less populated molten globule-like intermediate. The formation of the dominant non-local contact constitutes an example of the postulated Nature’s Shortcut to the prion protein collapse into the native structure. The possible role of the less populated molten globule-like intermediate is explored as the potential initiation point for the folding for three pathogenic mutants (T182A, I214V, and Q211P in mouse prion numbering) of the prion protein.

Apoptosis, as the major type of programmed cell death, plays an important role in the organism renewal and removal of defective and transformed cells, including cancer cells. One of the earliest apoptotic events is lipid peroxidation in the inner mitochondrial membrane catalyzed by a complex of cytochrome c (CytC) with the mitochondrial phospholipid cardiolipin (CL). It was shown that mixing CytC and CL solutions results in the formation of CytC/CL complexes (Cyt-CL nanospheres) with a diameter of 11–12 nm composed of the molten globule protein molecule and a CL monolayer. Using the methods of dynamic light scattering for the Cyt-CL chloroform solution and small-angle X-ray scattering for the Cyt-CL sediment, it was found that in both cases, Cyt-CL formed nanospheres with a diameter of 8 and 11 nm, which corresponded to the earlier determined lipid/protein ratios of 13–14 and 35–50, respectively. These results showed that the Cyt-CL nanospheres can form not only during crystallization but also in a hydrophobic medium. CytC in the complex exists as a molten globule, as evidenced by the emergence of tryptophan and tyrosine fluorescence (absent in the native protein) due to the Förster resonance transfer of the electron excitation energy onto the heme. At the same time, the coordinate bond between the heme iron and the sulfur atom of methionine 80 in Cyt-CL is disrupted (the absorption band at ~700 nm disappears). Similar disruption of the iron-sulfur bond in Cyt-CL was observed in 50% methanol. These changes were reversible, which corroborates the conclusion on the CytC transition to the molten globule conformation in methanol-containing solutions. [ABSTRACT FROM AUTHOR]

We have used computer simulations to investigate the structural nature of the molten globule (MG) state of canine milk lysozyme. To sample the conformational space efficiently, we performed replica‐exchange umbrella sampling simulations with the radius of gyration as a reaction coordinate. We applied the Weighted Histogram Analysis Method to the trajectory of the simulations to obtain the potential of mean force, from which we identified representative structures corresponding to local minima in the free energy surface. The representative structures obtained in this way are in accord with the characteristics of the MG state reported previously by experimental studies. We conjecture that the MG state comprises a series of partially structured states undergoing relatively fast conformational interchange. [ABSTRACT FROM AUTHOR]

Human thrombopoietin (hTPO) is a primary hematopoietic growth factor that regulates megakaryocytopoiesis and platelet production. The non‐glycosylated form of 1–163 residues of hTPO (hTPO163) including the N‐terminal active site domain (1–153 residues) is a candidate for treating thrombocytopenia. However, the autoantigenicity level of hTPO163 is higher than that of the full‐length glycosylated hTPO (ghTPO332). In order to clarify the structural and physicochemical properties of hTPO163, circular dichroism (CD) and differential scanning calorimetry (DSC) analyses were performed. CD analysis indicated that hTPO163 undergoes an induced‐fit conformational change (+19.0% for helix and −16.7% for β‐strand) upon binding to the neutralizing antibody TN1 in a manner similar to the coupled folding and binding mechanism. Moreover, DSC analysis showed that the thermal transition process of hTPO163 is a multistate transition; hTPO163 is thermally stabilized upon receptor (c‐Mpl) binding, as indicated with raising the midpoint (Tm) temperature of the transition by at least +9.5 K. The conformational variability and stability of hTPO163 indicate that hTPO163 exists as a molten globule under native conditions, which may enable the induced‐fit conformational change according to the type of ligands (antibodies and receptor). Additionally, CD and computational analyses indicated that the C‐terminal domain (154–332 residues) and glycosylation assists the folding of the N‐terminal domain. These observations suggest that the antibody affinity and autoantigenicity of hTPO163 might be reduced, if the conformational variability of hTPO163 is restricted by mutation and/or by the addition of C‐terminal domain with glycosylation to keep its conformation suitable for the c‐Mpl recognition. [ABSTRACT FROM AUTHOR]

β‐Cardiotoxin is a novel member of the snake venom three‐finger toxin (3FTX) family. This is the first exogenous protein to antagonize β‐adrenergic receptors and thereby causing reduction in heart rates (bradycardia) when administered into animals, unlike the conventional cardiotoxins as reported earlier. 3FTXs are stable all β‐sheet peptides with 60–80 amino acid residues. Here, we describe the three‐dimensional crystal structure of β‐cardiotoxin together with the identification of a molten globule intermediate in the unfolding pathway of this protein. In spite of the overall structural similarity of this protein with conventional cardiotoxins, there are notable differences observed at the loop region and in the charge distribution on the surface, which are known to be critical for cytolytic activity of cardiotoxins. The molten globule intermediate state present in the thermal unfolding pathway of β‐cardiotoxin was however not observed during the chemical denaturation of the protein. Interestingly, circular dichroism (CD) and NMR studies revealed the presence of α‐helical secondary structure in the molten globule intermediate. These results point to substantial conformational plasticity of β‐cardiotoxin, which might aid the protein in responding to the sometimes conflicting demands of structure, stability, and function during its biological lifetime. PDB Code(s): 3PLC; [ABSTRACT FROM AUTHOR]

Abstract Two novel medicinally important legume lectins from Bauhinia purpurea (BPL) and Wisteria floribunda (WFL) possessing extended sugar binding site were investigated for functional and conformational transitions using biochemical and biophysical techniques as well as bioinformatical tools. Homology model of BPL was constructed using the Schrodinger suite and docked with N-acetyl galactosamine and T-antigen disaccharide (Galβ1-3GalNAcαO-Me). The longer loop D in the structure of WFL compared to that in BPL was found to be responsible for its specificity to LacdiNac (β-D-GalNAc-[1 → 4]-DGlcNAc) over Galβ1-3GalNAc. BPL remained functionally stable up to 40 °C whereas WFL remained stable upto 70 °C indicating the strength of the sugar binding site geometry. Both the lectins showed intense but non-specific secondary structure in the range of 65–90 °C. WFL showed rapid aggregation above 80 °C as indicated by light scattering intensity. The lectins showed simultaneous dissociation and multistate unfolding in the vicinity of GdnHCl. At pH 1.0, both the lectins exhibited molten globule like structures, which were characterized further and were found to respond in a different way towards denaturants. The results have provided valuable insights into the molecular basis of the activity and stability of the two lectins. Highlights • Saccharide binding and structural transitions of Bauhinia and Wisteria lectins. • Homology model and docking of Bauhinia lectin. • Structural elements responsible for different disaccharide specificity. • Unique thermal transitions of the lectins. • Characterization of acid induced molten globule like species. [ABSTRACT FROM AUTHOR]

A systematic study on the self-assembled solution system of human serum albumin (HSA) and paramagnetic doxyl stearic acid (5-DSA and 16-DSA) ligands is reported covering the broad pH range 0.7-12.9, mainly using electron paramagnetic resonance (EPR) methods. It is tested to which extent the pH-induced conformational isomers of HSA reveal themselves in continuous wave (CW) EPR spectra from this spin probing approach in comparison to an established spin-labeling strategy utilizing 3-maleimido proxyl (5-MSL). Most analyses are conducted on empirical levels with robust strategies that allow for the detection of dynamic changes of ligand, as well as protein. Special emphasis has been placed on the EPR spectroscopic detection of a molten globule (MG) state of HSA that is typically found by the fluorescent probe 8-Anilino- naphthalene-1-sulfonic acid (ANS). Moreover, four-pulse double electron-electron resonance (DEER) experiments are conducted and substantiated with dynamic light scattering (DLS) data to determine changes in the solution shape of HSA with pH. All results are ultimately combined in a detailed scheme that describes the pH-induced functional phase space of HSA. [ABSTRACT FROM AUTHOR]

The majority of proteins perform their cellular function after folding into a specific and stable native structure. Additionally, for many proteins less compact ‘molten globule’ states have been observed. Current experimental observations show that the molten globule state can show varying degrees of compactness and solvent accessibility; the underlying molecular cause for this variation is not well understood. While the specificity of protein folding can be studied using protein lattice models, current design procedures for these models tend to generate sequences without molten globule-like behaviour. Here we alter the design process so the distance between the molten globule ensemble and the native structure can be steered; this allows us to design protein sequences with a wide range of folding pathways, and sequences with well-defined heat-induced molten globules. Simulating these sequences we find that (1) molten globule states are compact, but have less specific configurations compared to the folded state, (2) the nature of the molten globule state is highly sequence dependent, (3) both two-state and multi-state folding proteins may show heat-induced molten globule states, as observed in heat capacity curves. The varying nature of the molten globules and typical heat capacity curves associated with the transitions closely resemble experimental observations. [ABSTRACT FROM AUTHOR]